The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain.

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

Phenotypical, genotypical and pathological characterization of the moonwalker mouse, a model of ataxia.

We performed a comprehensive study of the morphological, functional, and genetic features of moonwalker (MWK) mice, a mouse model of spinocerebellar ataxia caused by a gain of function of the TRPC3 channel. These mice show numerous behavioral symptoms including tremor, altered gait, circling behavior, impaired motor coordination, impaired motor learning and decreased limb strength. Cerebellar pathology is characterized by early and almost complete loss of unipolar brush cells as well as slowly progressive, moderate loss of Purkinje cell (PCs). Structural damage also includes loss of synaptic contacts from parallel fibers, swollen ER structures, and degenerating axons. Interestingly, no obvious correlation was observed between PC loss and severity of the symptoms, as the phenotype stabilizes around 2 months of age, while the cerebellar pathology is progressive. This is probably due to the fact that PC function is severely impaired much earlier than the appearance of PC loss. Indeed, PC firing is already impaired in 3 weeks old mice. An interesting feature of the MWK pathology that still remains to be explained consists in a strong lobule selectivity of the PC loss, which is puzzling considering that TRPC is expressed in every PC. Intriguingly, genetic analysis of MWK cerebella shows, among other alterations, changes in the expression of both apoptosis inducing and resistance factors possibly suggesting that damaged PCs initiate specific cellular pathways that protect them from overt cell loss.

A novel role for the late-onset Alzheimer's disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling.

Postsynaptic trafficking plays a key role in regulating synapse structure and function. While spiny excitatory synapses can be stable throughout adult life, their morphology and function is impaired in Alzheimer's disease (AD). However, little is known about how AD risk genes impact synaptic function. Here we used structured superresolution illumination microscopy (SIM) to study the late-onset Alzheimer's disease (LOAD) risk factor BIN1, and show that this protein is abundant in postsynaptic compartments, including spines. While postsynaptic Bin1 shows colocalization with clathrin, a major endocytic protein, it also colocalizes with the small GTPases Rab11 and Arf6, components of the exocytic pathway. Bin1 participates in protein complexes with Arf6 and GluA1, and manipulations of Bin1 lead to changes in spine morphology, AMPA receptor surface expression and trafficking, and AMPA receptor-mediated synaptic transmission. Our data provide new insights into the mesoscale architecture of postsynaptic trafficking compartments and their regulation by a major LOAD risk factor.

Self-assembling vascular endothelial growth factor nanoparticles improve function in spinocerebellar ataxia type 1.

There is increasing appreciation for the role of the neurovascular unit in neurodegenerative diseases. We showed previously that the angiogenic and neurotrophic cytokine, vascular endothelial growth factor (VEGF), is suppressed to abnormally low levels in spinocerebellar ataxia type 1 (SCA1), and that replenishing VEGF reverses the cerebellar pathology in SCA1 mice. In that study, however, we used a recombinant VEGF, which is extremely costly to manufacture and biologically unstable as well as immunogenic. To develop a more viable therapy, here we test a synthetic VEGF peptide amphiphile that self-assembles into nanoparticles. We show that this nano-VEGF has potent neurotrophic and angiogenic properties, is well-tolerated, and leads to functional improvement in SCA1 mice even when administered at advanced stages of the disease. This approach can be generalized to other neurotrophic factors or molecules that act in a paracrine manner, offering a novel therapeutic strategy for neurodegenerative conditions.

Loss of M1 Receptor Dependent Cholinergic Excitation Contributes to mPFC Deactivation in Neuropathic Pain.

In chronic pain, the medial prefrontal cortex (mPFC) is deactivated and mPFC-dependent tasks such as attention and working memory are impaired. We investigated the mechanisms of mPFC deactivation in the rat spared nerve injury (SNI) model of neuropathic pain. Patch-clamp recordings in acute slices showed that, 1 week after the nerve injury, cholinergic modulation of layer 5 (L5) pyramidal neurons was severely impaired. In cells from sham-operated animals, focal application of acetylcholine induced a left shift of the input/output curve and persistent firing. Both of these effects were almost completely abolished in cells from SNI-operated rats. The cause of this impairment was an ∼60% reduction of an M1-coupled, pirenzepine-sensitive depolarizing current, which appeared to be, at least in part, the consequence of M1 receptor internalization. Although no changes were detected in total M1 protein or transcript, both the fraction of the M1 receptor in the synaptic plasma membrane and the biotinylated M1 protein associated with the total plasma membrane were decreased in L5 mPFC of SNI rats. The loss of excitatory cholinergic modulation may play a critical role in mPFC deactivation in neuropathic pain and underlie the mPFC-specific cognitive deficits that are comorbid with neuropathic pain.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) undergoes major reorganization in chronic pain. Deactivation of mPFC output is causally correlated with both the cognitive and the sensory component of neuropathic pain. Here, we show that cholinergic excitation of commissural layer 5 mPFC pyramidal neurons is abolished in neuropathic pain rats due to a severe reduction of a muscarinic depolarizing current and M1 receptor internalization. Therefore, in neuropathic pain rats, the acetylcholine (ACh)-dependent increase in neuronal excitability is reduced dramatically and the ACh-induced persisting firing, which is critical for working memory, is abolished. We propose that the blunted cholinergic excitability contributes to the functional mPFC deactivation that is causal for the pain phenotype and represents a cellular mechanism for the attention and memory impairments comorbid with chronic pain.

Activation of astrocytic PAR1 receptors in the rat nucleus of the solitary tract regulates breathing through modulation of presynaptic TRPV1.

KEY POINTS: In the rat nucleus of the solitary tract (NTS), activation of astrocytic proteinase-activated receptor 1 (PAR1) receptors leads to potentiation of neuronal synaptic activity by two mechanisms, one TRPV1-dependent and one TRPV1-independent. PAR1-dependent activation of presynaptic TRPV1 receptors facilitates glutamate release onto NTS neurons. The TRPV1-dependent mechanism appears to rely on astrocytic release of endovanilloid-like molecules. A subset of NTS neurons excited by PAR1 directly project to the rostral ventral respiratory group. The PAR1 initiated, TRPV1-dependent modulation of synaptic transmission in the NTS contributes to regulation of breathing. ABSTRACT: Many of the cellular and molecular mechanisms underlying astrocytic modulation of synaptic function remain poorly understood. Recent studies show that G-protein coupled receptor-mediated astrocyte activation modulates synaptic transmission in the nucleus of the solitary tract (NTS), a brainstem nucleus that regulates crucial physiological processes including cardiorespiratory activity. By using calcium imaging and patch clamp recordings in acute brain slices of wild-type and TRPV1-/- rats, we show that activation of proteinase-activated receptor 1 (PAR1) in NTS astrocytes potentiates presynaptic glutamate release on NTS neurons. This potentiation is mediated by both a TRPV1-dependent and a TRPV1-independent mechanism. The TRPV1-dependent mechanism appears to require release of endovanilloid-like molecules from astrocytes, which leads to subsequent potentiation of presynaptic glutamate release via activation of presynaptic TRPV1 channels. Activation of NTS astrocytic PAR1 receptors elicits cFOS expression in neurons that project to respiratory premotor neurons and inhibits respiratory activity in control, but not in TRPV1-/- rats. Thus, activation of astrocytic PAR1 receptor in the NTS leads to a TRPV1-dependent excitation of NTS neurons causing a potent modulation of respiratory motor output.

Absence of alsin function leads to corticospinal motor neuron vulnerability via novel disease mechanisms.

Mutations in the ALS2 gene result in early-onset amyotrophic lateral sclerosis, infantile-onset ascending hereditary spastic paraplegia and juvenile primary lateral sclerosis, suggesting prominent upper motor neuron involvement. However, the importance of alsin function for corticospinal motor neuron (CSMN) health and stability remains unknown. To date, four separate alsin knockout (Alsin(KO)) mouse models have been generated, and despite hopes of mimicking human pathology, none displayed profound motor function defects. This, however, does not rule out the possibility of neuronal defects within CSMN, which is not easy to detect in these mice. Detailed cellular analysis of CSMN has been hampered due to their limited numbers and the complex and heterogeneous structure of the cerebral cortex. In an effort to visualize CSMN in vivo and to investigate precise aspects of neuronal abnormalities in the absence of alsin function, we generated Alsin(KO)-UeGFP mice, by crossing Alsin(KO) and UCHL1-eGFP mice, a CSMN reporter line. We find that CSMN display vacuolated apical dendrites with increased autophagy, shrinkage of soma size and axonal pathology even in the pons region. Immunocytochemistry coupled with electron microscopy reveal that alsin is important for maintaining cellular cytoarchitecture and integrity of cellular organelles. In its absence, CSMN displays selective defects both in mitochondria and Golgi apparatus. UCHL1-eGFP mice help understand the underlying cellular factors that lead to CSMN vulnerability in diseases, and our findings reveal unique importance of alsin function for CSMN health and stability.

Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2.

Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyotrophic lateral sclerosis (ALS), frontotemporal type of dementia, or both. However, the molecular mechanisms are unknown. Here, we show that ALS/dementia-linked UBQLN2(P497H) transgenic mice develop neuronal pathology with ubiquilin2/ubiquitin/p62-positive inclusions in the brain, especially in the hippocampus, recapitulating several key pathological features of dementia observed in human patients with UBQLN2 mutations. A major feature of the ubiquilin2-related pathology in these mice, and reminiscent of human disease, is a dendritic spinopathy with protein aggregation in the dendritic spines and an associated decrease in dendritic spine density and synaptic dysfunction. Finally, we show that the protein inclusions in the dendritic spines are composed of several components of the proteasome machinery, including Ub(G76V)-GFP, a representative ubiquitinated protein substrate that is accumulated in the transgenic mice. Our data, therefore, directly link impaired protein degradation to inclusion formation that is associated with synaptic dysfunction and cognitive deficits. These data imply a convergent molecular pathway involving synaptic protein recycling that may also be involved in other neurodegenerative disorders, with implications for development of widely applicable rational therapeutics.

α-Synuclein expression in the mouse cerebellum is restricted to VGluT1 excitatory terminals and is enriched in unipolar brush cells.

α-Synuclein has a crucial role in synaptic vesicle release and synaptic membrane recycling. Although its general expression pattern has been described in the cerebellum, the precise cerebellar structures where α-synuclein is localized are poorly understood. To address this question, we used α-synuclein immunohistochemistry in adult mice cerebellar sections. We found that α-synuclein labels glutamatergic but not glycinergic and GABAergic synaptic terminals in the molecular and granule cell layers. α-Synuclein was preferentially expressed in parallel and mossy fiber synaptic terminals that also express vesicular glutamate transporter 1 (VGluT1), while it was not detected in VGluT2-positive climbing fibers. α-Synuclein was particularly enriched in lobules IX and X, a region known to contain a high density of unipolar brush cells (UBCs). To elucidate whether the α-synuclein-positive mossy fibers belong to UBCs, we double-labeled cerebellar sections with antibodies to α-synuclein and UBC-type-specific markers (calretinin for type I and metabotropic glutamate receptor 1α (mGluR1α) for type II UBCs) and took advantage of organotypic cerebellar cultures (in which all mossy fibers are UBC axons) and moonwalker mice (in which almost all UBCs are ablated) and found that both type I and type II UBCs express α-synuclein. In moonwalker mutant cerebella, the α-synuclein/VGluT1 immunolabeling showed a dramatic decrease in the vestibulocerebellum that correlated with the absence of UBC. α-Synuclein appears to be an excellent marker for intrinsic mossy fibers of the VGluT1 subset in conjunction with UBCs of both subtypes.

Temperature-sensitive Cav1.2 calcium channels support intrinsic firing of pyramidal neurons and provide a target for the treatment of febrile seizures.

Febrile seizures are associated with increased brain temperature and are often resistant to treatments with antiepileptic drugs, such as carbamazepine and phenytoin, which are sodium channel blockers. Although they are clearly correlated with the hyperthermic condition, the precise cellular mechanisms of febrile seizures remain unclear. We performed patch-clamp recordings from pyramidal cells in acute rat brain slices at temperatures up to 40°C and found that, at ≥37°C, L-type calcium channels are active at unexpectedly hyperpolarized potentials and drive intrinsic firing, which is also supported by a temperature-dependent, gadolinium-sensitive sodium conductance. Pharmacological data, RT-PCR, and the current persistence in Cav1.3 knock-out mice suggested a critical contribution of Cav1.2 subunits to the temperature-dependent intrinsic firing, which was blocked by nimodipine. Because intrinsic firing may play a critical role in febrile seizures, we tested the effect of nimodipine in an in vivo model of febrile seizures and found that this drug dramatically reduces both the incidence and duration of febrile seizures in rat pups, suggesting new possibilities of intervention for this important pathological condition.

Early onset of ataxia in moonwalker mice is accompanied by complete ablation of type II unipolar brush cells and Purkinje cell dysfunction.

Transient receptor potential "canonical" cation channels (TRPC) are involved in many cellular activities, including neuronal synaptic transmission. These channels couple lipid metabolism, calcium homeostasis, and electrophysiological properties as they are calcium permeable and activated through the phospholipase C pathway and by diacylglycerol. The TRPC3 subunit is abundantly expressed in Purkinje cells (PCs), where it mediates slow metabotropic glutamate receptor-mediated synaptic responses. Recently, it has been shown that heterozygous moonwalker mice, which are a model of cerebellar ataxia, carry a dominant gain-of-function mutation (T635A) in the TRPC3 gene. This mutation leads to PC loss and dysmorphism, which have been suggested to cause the ataxia. However, the ataxic phenotype is present from a very early stage (before weaning), whereas PC loss does not appear until several months of age. Here we show that another class of cerebellar neurons, the type II unipolar brush cells (UBCs), express functional TRPC3 channels; intriguingly, these cells are ablated in moonwalker mice by 1 month of age. Additionally, we show that in moonwalker mice, intrinsic excitability of PCs is altered as early as 3 weeks after birth. We suggest that this altered excitability and the TRPC3-mediated loss of type II UBCs may both contribute to the ataxic phenotype of these mice and that different calcium handling in PCs and type II UBCs may account for the dramatic differences in sensitivity to the moonwalker mutation between these cell types.

Expression of background potassium channels in rat DRG is cell-specific and down-regulated in a neuropathic pain model.

Neuropathic pain is associated with hyperexcitability of DRG neurons. Despite the importance of leakage potassium channels for neuronal excitability, little is known about their cell-specific expression in DRGs and possible modulation in neuropathic pain. Multiple leakage channels are expressed in DRG neurons, including TASK1, TASK3, TRESK, TRAAK, TWIK1, TREK1 and TREK2 but little is known about their distribution among different cell types. Our immunohistochemical studies show robust TWIK1 expression in large and medium size neurons, without overlap with TRPV1 or IB4 staining. TASK1 and TASK3, on the contrary, are selectively expressed in small cells; TASK1 expression closely overlaps TRPV1-positive cells, while TASK3 is expressed in TRPV1- and IB4-negative cells. We also studied mRNA expression of these channels in L4-L5 DRGs in control conditions and up to 4 weeks after spared nerve injury lesion. We found that TWIK1 expression is much higher than TASK1 and TASK3 and is strongly decreased 1, 2 and 4 weeks after neuropathic injury. TASK3 expression, on the other hand, decreases 1 week after surgery but reverts to baseline by 2weeks; TASK1 shows no significant change at any time point. These data suggest an involvement of TWIK1 in the maintenance of the pain condition.

Bidirectional Regulation of GABAA Reversal Potential in the Adult Brain: Physiological and Pathological Implications.

In physiological conditions, the intracellular chloride concentration is much lower than the extracellular. As GABAA channels are permeable to anions, the reversal potential of GABAA is very close to that of Cl-, which is the most abundant free anion in the intra- and extracellular spaces. Intracellular chloride is regulated by the activity ratio of NKCC1 and KCC2, two chloride-cation cotransporters that import and export Cl-, respectively. Due to the closeness between GABAA reversal potential and the value of the resting membrane potential in most neurons, small changes in intracellular chloride have a major functional impact, which makes GABAA a uniquely flexible signaling system. In most neurons of the adult brain, the GABAA reversal potential is slightly more negative than the resting membrane potential, which makes GABAA hyperpolarizing. Alterations in GABAA reversal potential are a common feature in numerous conditions as they are the consequence of an imbalance in the NKCC1-KCC2 activity ratio. In most conditions (including Alzheimer's disease, schizophrenia, and Down's syndrome), GABAA becomes depolarizing, which causes network desynchronization and behavioral impairment. In other conditions (neonatal inflammation and neuropathic pain), however, GABAA reversal potential becomes hypernegative, which affects behavior through a potent circuit deactivation.

Hypernegative GABAA Reversal Potential in Pyramidal Cells Contributes to Medial Prefrontal Cortex Deactivation in a Mouse Model of Neuropathic Pain.

Deactivation of the medial prefrontal cortex (mPFC) has been broadly reported in both neuropathic pain models and human chronic pain patients. Several cellular mechanisms may contribute to the inhibition of mPFC activity, including enhanced GABAergic inhibition. The functional effect of GABAA(γ-aminobutyric acid type A)-receptor activation depends on the concentration of intracellular chloride in the postsynaptic neuron, which is mainly regulated by the activity of Na-K-2Cl cotransporter isoform 1 (NKCC1) and K-Cl cotransporter isoform 2 (KCC2), 2 potassium-chloride cotransporters that import and extrude chloride, respectively. Recent work has shown that the NKCC1-KCC2 ratio is affected in numerous pathological conditions, and we hypothesized that it may contribute to the alteration of mPFC function in neuropathic pain. We used quantitative in situ hybridization to assess the level of expression of NKCC1 and KCC2 in the mPFC of a mouse model of neuropathic pain (spared nerve injury), and we found that KCC2 transcript is increased in the mPFC of spared nerve injury mice while NKCC1 is not affected. Perforated patch recordings further showed that this results in the hypernegative reversal potential of the GABAA current in pyramidal neurons of the mPFC. Computational simulations suggested that this change in GABAA reversal potential is sufficient to significantly reduce the overall activity of the cortical network. Thus, our results identify a novel pathological modulation of GABAA function and a new mechanism by which mPFC function is inhibited in neuropathic pain. Our data also help explain previous findings showing that activation of mPFC interneurons has proalgesic effect in neuropathic, but not in control conditions. PERSPECTIVE: Chronic pain is associated with the presence of depolarizing GABAA current in the spinal cord, suggesting that pharmacological NKCC1 antagonism has analgesic effects. However, our results show that in neuropathic pain, GABAA current is actually hyperinhibitory in the mPFC, where it contributes to the mPFC functional deactivation. This suggests caution in the use of NKCC1 antagonism to treat pain.

Abnormalities in hippocampal functioning with persistent pain.

Chronic pain patients exhibit increased anxiety, depression, and deficits in learning and memory. Yet how persistent pain affects the key brain area regulating these behaviors, the hippocampus, has remained minimally explored. In this study we investigated the impact of spared nerve injury (SNI) neuropathic pain in mice on hippocampal-dependent behavior and underlying cellular and molecular changes. In parallel, we measured the hippocampal volume of three groups of chronic pain patients. We found that SNI animals were unable to extinguish contextual fear and showed increased anxiety-like behavior. Additionally, SNI mice compared with Sham animals exhibited hippocampal (1) reduced extracellular signal-regulated kinase expression and phosphorylation, (2) decreased neurogenesis, and (3) altered short-term synaptic plasticity. To relate the observed hippocampal abnormalities with human chronic pain, we measured the volume of human hippocampus in chronic back pain (CBP), complex regional pain syndrome (CRPS), and osteoarthritis patients (OA). Compared with controls, CBP and CRPS, but not OA, had significantly less bilateral hippocampal volume. These results indicate that hippocampus-mediated behavior, synaptic plasticity, and neurogenesis are abnormal in neuropathic rodents. The changes may be related to the reduction in hippocampal volume we see in chronic pain patients, and these abnormalities may underlie learning and emotional deficits commonly observed in such patients.

pH modulation of glial glutamate transporters regulates synaptic transmission in the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is the major site for termination of visceral sensory afferents contributing to homeostatic regulation of, for example, arterial pressure, gastric motility, and breathing. Whereas much is known about how different neuronal populations influence these functions, information about the role of glia remains scant. In this article, we propose that glia may contribute to NTS functions by modulating excitatory neurotransmission. We found that acidification (pH 7.0) depolarizes NTS glia by inhibiting K(+)-selective membrane currents. NTS glia also showed functional expression of voltage-sensitive glutamate transporters, suggesting that extracellular acidification regulates synaptic transmission by compromising glial glutamate uptake. To test this hypothesis, we evoked glutamatergic slow excitatory potentials (SEPs) in NTS neurons with repetitive stimulation (20 pulses at 10 Hz) of the solitary tract. This SEP depends on accumulation of glutamate following repetitive stimulation, since it was potentiated by blocking glutamate uptake with dl-threo-ß-benzyloxyaspartic acid (TBOA) or a glia-specific glutamate transport blocker, dihydrokainate (DHK). Importantly, extracellular acidification (pH 7.0) also potentiated the SEP. This effect appeared to be mediated through a depolarization-induced inhibition of glial transporter activity, because it was occluded by TBOA and DHK. In agreement, pH 7.0 did not directly alter d-aspartate-induced responses in NTS glia or properties of presynaptic glutamate release. Thus acidification-dependent regulation of glial function affects synaptic transmission within the NTS. These results suggest that glia play a modulatory role in the NTS by integrating local tissue signals (such as pH) with synaptic inputs from peripheral afferents.

Impaired muscarinic modulation of the rat prelimbic cortex in neuropathic pain is sexually dimorphic and associated with cold allodynia.

Cholinergic modulation of the brain cortex is critical for cognitive processes, and altered cholinergic modulation of the prefrontal cortex is emerging as an important mechanism of neuropathic pain. Sex differences in pain prevalence and perception are well known, yet the precise nature of the mechanisms responsible for sexual dimorphism in chronic neuropathic pain are poorly understood. Here we investigated potential sex differences in cholinergic modulation of layer five commissural pyramidal neurons of the rat prelimbic cortex in control conditions and in the SNI model of neuropathic pain. We discovered that cholinergic modulation is stronger in cells from male compared with female rats, and that in neuropathic pain rats, cholinergic excitation of pyramidal neurons was more severely impaired in males than in females. Finally, we found that selective pharmacological blockade of the muscarinic M1 subunit in the prefrontal cortex induces cold sensitivity (but not mechanical allodynia) in naïve animals of both sexes.

Acid-sensing ion channels contribute to chemosensitivity of breathing-related neurons of the nucleus of the solitary tract.

Cellular mechanisms of central pH chemosensitivity remain largely unknown. The nucleus of the solitary tract (NTS) integrates peripheral afferents with central pathways controlling breathing; NTS neurons function as central chemosensors, but only limited information exists concerning the ionic mechanisms involved. Acid-sensing ion channels (ASICs) mediate chemosensitivity in nociceptive terminals, where pH values ∼6.5 are not uncommon in inflammation, but are also abundantly expressed throughout the brain where pHi s tightly regulated and their role is less clear. Here we test the hypothesis that ASICs are expressed in NTS neurons and contribute to intrinsic chemosensitivity and control of breathing. In electrophysiological recordings from acute rat NTS slices, ∼40% of NTS neurons responded to physiological acidification (pH 7.0) with a transient depolarization. This response was also present in dissociated neurons suggesting an intrinsic mechanism. In voltage clamp recordings in slices, a pH drop from 7.4 to 7.0 induced ASIC-like inward currents (blocked by 100 µM amiloride) in ∼40% of NTS neurons, while at pH ≤ 6.5 these currents were detected in all neurons tested; RT-PCR revealed expression of ASIC1 and, less abundantly, ASIC2 in the NTS. Anatomical analysis of dye-filled neurons showed that ASIC-dependent chemosensitive cells (cells responding to pH 7.0) cluster dorsally in the NTS. Using in vivo retrograde labelling from the ventral respiratory column, 90% (9/10) of the labelled neurons showed an ASIC-like response to pH 7.0, suggesting that ASIC currents contribute to control of breathing. Accordingly, amiloride injection into the NTS reduced phrenic nerve activity of anaesthetized rats with an elevated arterial P(CO(2)) .

Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies.

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.

Electrophysiological, morphological, and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells.

Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC transgenic mice, which selectively express enhanced green fluorescent protein (EGFP) in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells, EGFP-positive UBCs had smaller somata (area 48 vs 63 µm(2)), lower specific membrane resistance (6.4 vs. 13.7 KΩ cm(2)), were less prone to intrinsic firing, and showed more irregular firing (in cell-attached ~49 % were firing vs. ~88 %, and the CV was 0.53 vs. 0.32 for EGFP-negative cells). Some of these differences are attributable to higher density of background K(+) currents in EGFP-positive cells (at -120 mV, the barium-sensitive current was 94 vs. 37 pA in EGFP-negative cells); Ih, on the contrary, was more abundantly expressed in EGFP-negative cells (at -140 mV, it was -122 vs. -54 pA in EGFP-positive neurons); furthermore, while group II mGluR modulation of the background potassium current in EGFP-negative UBCs was maintained after intracellular dialysis, mGluR modulation in EGFP-positive UBCs was lost in whole-cell recordings. Finally, cell-attached firing was reversibly abolished by the GABA(B) activation in EGFP-positive, but not in EGFP-negative UBCs. Immunohistochemistry showed that EGFP-negative UBCs express GIRK2 at high density, while mGluR1α UBCs are GIRK2 negative, suggesting that GIRK2 mediates the mGluR-sensitive current in EGFP-negative UBCs. These data suggest that the two subclasses perform different functions in the cerebellar microcircuits.